Despite the recent surge of interest in malaria, Plasmodium vivax has remained relatively neglected compared to falciparum malaria. Over 2.5 billion persons are at risk of vivax infection, which causes between 80 and 300 million cases per year. The principal drug in use today for vivax malaria is chloroquine (CQ) but its efficacy in now threatened by evolving resistance. Over the past 20 years, CQ resistance emerged in Western Pacifica and Southeast (SE) Asia and is now spreading worldwide. In some countries, such as Cambodia and Indonesia, the diminished efficacy of CQ therapy has prompted a change to artemisinin combination therapies (ACTs), which are significantly more costly, as first-line therapy for vivax malaria. The molecular basis of P. vivax CQ resistance remains unknown. Evidence indicates that the genetic events underlying P. vivax CQ resistance differs from P. falciparum as no association was found between in vivo drug responses and codon changes in Plasmodium vivax chloroquine resistance transporter (pvcrt), the P. vivax ortholog of Plasmodium falciparum chloroquine resistance transporter (pfcrt). Further research on CQ-resistant P. vivax has been severely hampered by the lack of critical tools to study the parasites including in vitro culture, DNA transfection and cloning methods. Therefore research on P. vivax CQ resistance depends on non-human primate models. Recently, the National Institutes of Health (NIH) invested significant resources to generate the first P. vivax genetic cross between a CQ sensitive strain and a CQ resistant strain in a chimpanzee. Using this cross, our proposal is aimed at determining the genetic loci underlying CQ resistance in P. vivax by conducting a linkage group selection (LGS) analysis leveraging whole genome sequencing of parental strains and in vivo mixed progeny infections before and after drug pressure. Without the need for traditional clonal analysis and defined IC50s, LGS identifies shifts toward resistant parental alleles after selection with CQ indicative of genetic loci associated with CQ resistance and has been used to successfully identify resistance alleles/mutations multiple times in malaria rodent models. This proposal is clearly significant for multiple reasons: 1) CQ resistant vivax is a major global public health problem causing significant morbidity and mortality with significant financial implications, 2) this will be the first genome-wide assessment for genetic loci of CQ resistance in vivax malaria using a defined genetic cross, 3) it leverages a set of samples from a unique and irreplaceable genetic cross generated in a chimpanzee, the approach which identified the key P. falciparum mutations for resistance to CQ and other antimalarials, 4) identification of CQ resistance mechanisms will aid in the development of additional antimalarials, and 5) identification of loci associated with CQ resistance will provide molecular markers of resistance, an important public health tool for studying the spread of resistance. The proposal is also innovative as it leverages new tools for conducting whole genome sequencing using hybrid capture to enrich parasite DNA and remove contaminating host DNA. Understanding the molecular basis for CQ resistance in P. vivax would be a major advance in understanding of vivax biology and has the potential to have profound impacts on global public health.